GST pull-down

Approximately 5 µg of GST or GST-fusion proteins were incubated in Glutathione-Sepharose beads (GE Healthcare) in the GST-lysis buffer at 4°C overnight. The bead-bound proteins were then incubated in GST-lysis buffer containing 5% BSA at 4°C for 2 hours. Meanwhile, His-tagged recombinants (~50 µg) were pre-cleared in Glutathione-Sepharose beads. The pre-cleared protein was then incubated with bead-bound proteins in phosphate-buffered saline containing 0.1%

Tween20 (PBST). After 2 hours of incubation, the beads were washed three times with PBST containing 500 mM KCl and eluted with a 2x-SDS loading buffer. The pull-downs were then analyzed by SDS-PAGE and subsequent Coomassie staining. For specific detection of His-tagged recombinants, anti-His (1:1,000) (Abcam) was used. Bound primary antibodies were detected using Goat anti-Mouse IRDye (1:10,000) using an Odyssey imaging system (Li-Cor).

111

Chapter 4: Enhancement of microRNA-mediated deadenylation by the TRIM-NHL protein TRIM-NHL-2 and DEAD-box protein CGH-1

Vinay K. Mayya, Mathieu N. Flamand, Elva Vidya, Nahum Sonenberg, James A. Wohlschlegel, Christopher M. Hammell, Thomas F. Duchaine^*

Manuscript in preparation

*Correspondence: Thomas.duchaine@mcgill.ca

112 4.1 Abstract

The C. elegans NHL-2, a member of the broadly conserved TRIM-NHL family of RNA-binding proteins, enhances post-transcriptional repression of a subset of miRNA targets. Here, a co-immunoprecipitation and proteomic survey on NHL-2 detected interactions with components of miRISC, processing bodies (P-bodies), the CCR4-NOT deadenylase complex, and other RNA-binding proteins. Recruitment of NHL-2 to the 3`UTR of mRNAs triggered weak mRNA deadenylation, but this activity was greatly potentiated by juxtaposition to miRNA-binding sites.

CRISPR-Cas9 engineering of 3`UTR sequences in vivo further supported the function of enhancement of miRNA-mediated silencing in vivo. Finally, we demonstrate that NHL-2 directly associates with the DEAD-box RNA helicase CGH-1 (C. elegans ortholog of DDX6) in a temperature-dependent manner to support a model where the NHL-2/CGH-1 interaction potentiates miRNA-mediated deadenylation by remodeling the RNP. Altogether our findings shed light on the mechanistic basis of cooperativity between miRNAs, and the RNA-binding protein NHL-2.

Keywords: microRNA, NHL-2, CGH-1, cooperativity, deadenylation, P-bodies.

113 4.2 Introduction

Post-transcriptional regulation of gene expression is often mediated through the recognition of cis-regulatory sequences and structures in the 3`UTRs of mRNAs by trans-acting factors, some of which involve microRNA Induced Silencing Complex (miRISC), their associated machinery, and RNA-binding proteins (RBPs) (Mayya and Duchaine, 2019). Trans-acting factors, in turn, recruit effector proteins such as CCR4-NOT deadenylase, the decapping complex, and thus mediate gene repression (Duchaine and Fabian, 2019; Jonas and Izaurralde, 2015).

Considering the vast number of binding sites for miRNAs in 3`UTRs, multiple miRNAs can bind within a 3`UTR and alter mRNAs' fate through cooperative or competitive interplay (Friedman et al., 2009). For instance, proper neuronal patterning in C. elegans requires the repression of cog-1 mRNA through the cooperative action of two lsy-6 miRNA-binding sites in its 3` UTR (Didiano and Hobert, 2008; Johnston and Hobert, 2003). Recent in vitro and in vivo studies have provided more clarity on the mechanism underlying cooperativity. The presence of two or more miRNA-binding sites on target mRNAs can enable the recruitment of deadenylase complex to provide robust gene silencing (Flamand et al., 2017; Wu et al., 2010). Likewise, RBPs and miRNAs interactions within a 3`UTR are also thought to function cooperatively (Jiang and Coller, 2012).

Examples of a positive interplay between RBP and miRNAs include the cooperation of TTP with miR-16 in regulating TNF-alpha mRNA, and the modulation of local 3`UTR structures in c-Myc mRNA by HuR that increases let-7 miRNA activity (Jing et al., 2005; Kim et al., 2009). The mechanisms behind miRNA-RBP interactions occurring on 3`UTR of mRNAs remain poorly understood.

The broadly conserved Tripartite motif (TRIM)- NCL-1/H2TA/LIN-41 (NHL) family of RBPs are important regulators of development, cell polarity, and sex determination (Tocchini and Ciosk,

114 2015). The N-terminal TRIM contains three main domains: a RING finger, two B-Box-type zinc fingers, and a coiled-coil domain that can participate in protein-protein interactions and in some cases may act as an E3 ubiquitin ligase. The C-terminus consists of the NHL domain, which comprises five to six repeats of ~40 amino acids that fold into a β-propeller structure to bind directly to RNA (Davis et al., 2018; Loedige et al., 2015; Loedige et al., 2014). A subset of TRIM-NHL RBPs are implicated in post-transcriptional gene regulation. For instance, the Drosophila Brat recruits the effector protein 4EHP to repress the translation of hunchback mRNA (Cho et al., 2006). In C. elegans, nhl-2 is required to efficiently repress cog-1 and hbl-1 mRNAs by lsy-6 and let-7 miRNAs, respectively (Hammell et al., 2009b). More recently, in vitro binding assays using the NHL domain of NHL-2 revealed a strong binding preference for poly(U)-enriched RNAs (Davis et al., 2018). The endogenous targets and the exact mechanistic roles of NHL-2 in post-transcriptional gene repression are unclear.

Translationally repressed mRNAs and components of the miRISC and its associated machinery localize to P-bodies (Ding et al., 2005; Eystathioy et al., 2003; Liu et al., 2005). P-bodies are cytoplasmic membrane-less ribonucleoprotein (RNP) granules suspected to be sites for mRNA degradation and/or mRNA storage. An essential protein required for the assembly of RNPs is the conserved RNA dependent DEAD-box ATPase DDX6/RCK/Me31B/Dhh1/CGH-1 (Cougot et al., 2004; Eulalio et al., 2007b; Minshall et al., 2009; Serman et al., 2007; Sheth and Parker, 2003).

Results from several groups show that DDX6 and its orthologs also enhance translation repression (Chu and Rana, 2006; Coller and Parker, 2005; Minshall et al., 2009). For instance, DDX6 interacts directly with CNOT1 subunit of the CCR4-NOT deadenylase complex to repress miRNA targets (Chen et al., 2014; Mathys et al., 2014; Rouya et al., 2014). In C. elegans, CGH-1 binds and

115 stabilizes specific maternal transcripts during oogenesis (Boag et al., 2008). CGH-1 also colocalizes with NHL-2 in somatic cells in vivo (Hammell et al., 2009b).

Here, we uncovered the interactome of NHL-2 and identified extensive interactions with core components of miRISC, the CCR4-NOT deadenylase complex, P-bodies, and other RBPs. We show that tethering NHL-2 protein, in vitro and in vivo, greatly enhances the potency of miRNA-mediated deadenylation of target mRNAs. We further show a direct interaction between NHL-2 and the RNA helicase, CGH-1, and abolishing this interaction exacerbated miRNA defects. We propose a model wherein reorganization of RNP through CGH-1 and NHL-2 interaction promotes miRNA-mediated deadenylation.

116 4.3 Results

4.3.1 NHL-2 interacts with miRISC, CCR4-NOT complex, and the P-Body associated